This invention relates to methods of using human dendritic cells to present antigens for the induction of antigen-specific T cell-mediated immune responses. In particular, it relates to the isolation of dendritic cells from human blood, exposing the cells to antigens, co-culturing the antigen-pulsed dendritic cells with xcex3xcex4-T cell receptor-positive-T cells (xcex3xcex4-TCR+ T cells) obtained from unprimed or weakly primed individuals for the stimulation of antigen-specific T cell proliferative and cytotoxic activities. The dendritic cell antigen presentation system described herein has a wide range of applications, including but not limited to, activation and expansion of large numbers of antigen-specific major histocompatibility complex-unrestricted T cells for use in adoptive cellular immunotherapy against infectious diseases and cancer.
The introduction of a foreign antigen into an individual elicits an immune response consisting of two major components, the cellular and humoral immune responses, mediated by two functionally distinct populations of lymphocytes known as T and B cells, respectively. The T cells nay be further divided into two subsets by function and phenotype. A subset of T cells responds to antigen stimulation by producing lymphokines which xe2x80x9chelpxe2x80x9d or activate various other cell types in the immune system. Another T cell subset is capable of developing into antigen-specific cytotoxic effector cells, being able to directly kill antigen-positive target cells. On the other hand, the B cell response is primarily carried out by secretory proteins, antibodies, which directly bind and neutralize antigens.
Helper T cells (TH) can be distinguished from classical cytotoxic T lymphocytes (CTL) and B cells by their cell surface expression of a glycoprotein marker termed CD4. Although the mechanism by which CD4+ TH function has not been fully elucidated, the existence of functionally distinct subsets within the CD4+ T cell compartment has been reported (Mosmann and Coffman, 1989, Ann. Rev. Immunol. 7:145-173). In the mouse, type 1 helper T cells (TH1) produce interleukin-2 (IL-2) and xcex3-interferon (xcex3-IFN) upon activation, while type 2 helper T cells (TH2) produce IL-4 and IL-5. Based on the profile of lymphokine production, TH1 appear to be involved in promoting the activation and proliferation of other T cell subsets including CTL, whereas TH2 specifically regulate B cell proliferation and differentiation, antibody synthesis, and antibody class switching. Some CD4+ T cells, like CD8+ CTL, appear to be capable of cytotoxic effector function.
A second T cell subpopulation is the classical CTL which express the CD8 surface marker. Unlike most TH, these cells display cytolytic activity upon direct contact with target cells, although they are also capable of producing certain lymphokines. In vivo, CTL function is particularly important in situations where an antibody response alone is inadequate. There is a preponderance of experimental evidence that CTL rather than B cells and their antibody products play a principal role in the defense against viral infections and cancer.
A salient feature of both T and B cell responses is their exquisite specificity for the immunizing antigen; however, the mechanisms for antigen recognition differ between these two cell types. B cells recognize antigens by antibodies, either acting as cell surface receptors or as secreted proteins, which bind directly to antigens on a solid surface or in solution, whereas T cells only recognize antigens that have been processed or degraded into small fragments and presented on a solid phase such as the surface of antigen-presenting cells (APC). Additionally, antigenic fragments must be presented to T cells in association with major histocompatibility complex (MHC)-encoded class I or class II molecules. The MHC refers to a cluster of genes that encode proteins with diverse immunological functions. In man, the MHC is known as HLA. Class I gene products are found on all somatic cells, and they were originally discovered as targets of major transplantation rejection responses. Class II gene products are mostly expressed on cells of various hematopoietic lineages, and they are involved in cell-cell interactions in the immune system. Most importantly, MHC-encoded proteins have been shown to function as receptors for processed antigenic fragments on the surface of APC (Bjorkman et al., 1987, Nature 329: 506-512).
Another level of complexity in the interaction between a T cell expressing an xcex1xcex2-T cell receptor and an antigenic fragment is that it occurs only if the MHC molecules involved are the same on the APC and the responding T cells. In other words, a T cell specific for a particular antigenic epitope expresses a receptor having low affinity for self MHC proteins, which when such MHC proteins on APC are occupied by the epitope, engage the T cell in a stronger interaction leading to antigen-specific T cell activation. The phenomenon of a T cell reacting with a processed antigen only when presented by cells expressing a matching MHC is known as MHC-restriction. This requirement presents a practical limitation to the use of MHC-restricted T cells in cellular immunotherapy since the T cells must be matched at the MHC with a recipient""s target cells for them to be effective.
The specificity of T cell immune responses for antigens is a function of the unique receptors expressed by these cells. The T cell receptor (TCR) is structurally homologous to an antibody; it is a heterodimer composed of disulfide-linked glycoproteins. Four TCR polypeptide chains known as xcex1, xcex2, xcex3, and xcex4 have been identified, although the vast majority of functional T cells including both CD4+ TH and CD8+ CTL, express the xcex1xcex2 heterodimeric TCR. Transfer of xcex1 and xcex2 genes alone into recipient cells was shown to be both necessary and sufficient to confer antigen specificity and MHC-restriction (Dembic et al., 1986, Nature 320: 232-238). Thus, the xcex1xcex2 TCR appears to be responsible for recognizing a combination of antigenic fragment and MHC determinants. In this regard, the ability of an antibody specific for MHC class I or class II molecules to inhibit the antigen reactivity of a particular T cell population is often used as an indication that the T cells express xcex1xcex2-TCR.
The apparent basis of MHC restriction is that CD4+ T cells express xcex1xcex2 TCR which recognize antigenic fragments physically associated with MHC class II proteins, while the TCR on CD8+ CTL recognize MHC class I-associated fragments. Thus, CD4+ T cells can recognize only a restricted class of APC that are class II+, whereas CD8+ CTL can interact with virtually any antigen-positive cells, since all somatic cells express class I molecules. CD4+ CTL have been identified, and they are MHC class II restricted, and lyse target cells only if the latter express self-MHC class II determinants associated with specific antigenic fragments. Both CD4 and CD8 molecules also contribute to this interaction by binding to monotypic determinants on the MHC class II and I molecules, respectively.
A second type of TCR composed of xcex3xcex4 heterodimers is expressed by a small percentage of T cells. Approximately 10% of T cells in the peripheral blood express the xcex3xcex4-TCR, and a larger percentage of T cells in certain epithelial tissues such as the gut and skin are reported to be xcex3xcex4-positive (Allison and Havran, 1991, Annu. Rev. Immunol. 9:679-705). Although xcex3xcex4-TCR+ T cells differentiate in the thymus and appear to be able to respond to foreign antigens in a manner analogous to xcex1xcex2-TCR-bearing T cells, the physiologic role of xcex3xcex4-T cells is poorly understood. Some studies have shown that functionally active xcex3xcex4-T cells can be cytolytic in an MHC unrestricted manner. If so, it may be possible to utilize antigen-specific xcex3xcex4-TCR+ T cells generated from one individual to treat another individual who is not MHC compatible with the donor. However, since there is a relatively small number of germ line gene segments encoding the xcex3xcex4-TCR, the functional repertoire of xcex3xcex4-T cells is unknown. The xcex3xcex4-T cells are often referred to as being double negative because they lack the expression of CD4 and CD8 markers.
In summary, the generation of an immune response begins with the sensitization of CD4+ and CD8+ T cell subsets through their interaction with APC that express MHC-class I or class II molecules associated with antigenic fragments. The sensitized or primed CD4+ T cells produce lymphokines that participate in the activation of B cells as well as various T cell subsets. The sensitized CD8+ T cells increase in numbers in response to lymphokines and are capable of destroying any cells that express the specific antigenic fragments associated with matching MHC-encoded class I molecules. For example, in the course of a viral infection, CTL eradicate virally-infected cells, thereby limiting the progression of virus spread and disease development.
The presentation of antigens to T cells is carried out by specialized cell populations referred to as antigen presenting cells (APC). Typically, APC include macrophages/monocytes, B cells, and bone marrow-derived dendritic cells (DC). DC are sometimes also referred to as xe2x80x9cprofessionalxe2x80x9d APC. APC are capable of internalizing exogenous antigens, cleaving them into smaller fragments in enzyme-rich vesicles, and coupling the fragments to MHC-encoded class I or class II products for expression on the cell surface (Goldberg and Rock, 1992, Nature 357:375-379). Since APC express both MHC-encoded class I and class II glycoproteins, they can present antigenic fragments to both CD4+ and CD8+ T cells for the initiation of an immune response.
By definition, APC not only can present antigens to T cells with antigen-specific receptors, but can provide all the signals necessary for T cell activation. Such signals are incompletely defined, but probably involve a variety of cell surface molecules as well as cytokines or growth factors. Further, the factors necessary for the activation of naive or unprimed T cells may be different from those required for the re-activation of previously primed memory T cells. The ability of APC to both present antigens and deliver signals for T cell activation is commonly referred to as an accessory cell function. Although monocytes and B cells have been shown to be competent APC, their antigen presenting capacities in vitro appear to be limited to the re-activation of previously sensitized T cells. Hence, they are not capable of directly activating functionally naive or unprimed T cell populations.
Although it had been known for a long time that APC process and present antigens to T cells, it was not shown until relatively recently that small antigenic peptides could directly bind to MHC-encoded molecules (Babbit et al., 1985, Nature 317: 359; Townsend et al., 1986, Cell 44: 959). However, it is believed that normally, complex antigens are proteolytically processed into fragments inside the APC, and become physically associated with the MHC-encoded proteins intracellularly prior to trafficking to the cell surface as complexes. Two distinct pathways for antigen presentation have been proposed (Braciale et al., 1987, Immunol. Rev. 98: 95-114). It was thought that exogenous antigens were taken up by APC, processed and presented by the exogenous pathway to class II restricted CD4+ T cells, while the endogenous pathway processed intracellularly synthesized proteins, such as products of viral genes in virally-infected cells, for association with MHC class I proteins and presentation to CD8+ CTL. However, although the two pathways in antigen processing and presentation may still be correct in some respects, the distinction is blurred in light of recent findings that exogenously added antigens may also be presented to class I-restricted CTL (Moore et al., 1988, Cell 54: 777). Since most studies of antigen presentation and T cell activation have utilized xcex1xcex2-TCR+ T cells, it is still not known how xcex3xcex4-TCR+ T cells recognize and respond to antigens, especially in light of the finding that they may react with antigens in an MHC-unrestricted manner. Nor is it clear whether only certain types of APC are capable of presenting antigens to xcex3xcex4-T cells and whether naive unprimed xcex3xcex4-T cells can be activated by antigen-pulsed APC in vitro.
The term xe2x80x9cdendritic cellsxe2x80x9d refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues (Steinman, 1991, Ann. Rev. Immunol. 9:271-296). These cells include lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation. Although they are collectively classified as a group based on their morphology, high levels of surface MHC class II expression, and absence of certain other surface markers expressed on T cells, B cells, monocytes, and natural killer cells, it is presently not known whether they derive from a common precursor or can all function as APC in the same manner. Further, since the vast majority of published reports have utilized DC isolated from the mouse spleen, results from these studies may not necessarily correlate with the function of DC obtained from other tissue types. (Inaba et al., 1987, J. Exp. Med. 166:182-194; Hengel et al., 1987 J. Immunol., 139:4196-4202; Kast et al., 1988, J. Immunol., 140:3186-3193; Romani et al., 1989, J. Exp. Med. 169:1169-1178; Macatonia et al., 1989, J. Exp. Med. 169:1255-1264; Inaba et al., 1990, J. Exp. Med. 172:631-6640). For example, despite high levels of MHC-class II expression, mouse epidermal Langerhans cells, unlike splenic DC, are not active APC in mixed leucocyte reaction (MLR), unless cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) (Witmer-Pock et al., 1987, J. Exp. Med. 166:1484-1498; Heufler et al., 1988, J. Exp. Med. 167:700-705). Most human Langerhans cells express the CD1 and CD4 markers, while freshly isolated blood DC express CD4 weakly, but not CD1. On the other hand, cultured peripheral blood DC express CD1c, but not CD4. Additionally, it has not been established the extent to which the functional characteristics observed with mouse DC are applicable to human DC, especially the DC obtained from non-splenic tissues; in part, due to inherent differences between the human and murine immune systems.
Recently, a few studies have described the isolation of human DC from the peripheral blood. (Young and Steinman, 1990, J. Exp. Med. 171:1315-1332; Freudenthal and Steinman, 1990, Proc. Natl. Acad. Sci. USA 87:7698-7702; Macatonia et al., 1989, Immunol. 67:285-289; Markowicz and Engleman, 1990, J. Clin. Invest. 85:955-961). However, all reported isolation procedures invariably involve the use of sheep red blood cells and/or fetal calf serum, which are potentially immunogenic foreign antigens that can be presented by DC to T cells, and if so, would interfere with the antigen-specific responses desired. Most importantly, it has not been determined prior to Applicants"" invention whether human DC can, in fact, present exogenous antigens to xcex3xcex4-TCR+ T cells because human DC have only been tested as APC for xcex1xcex2-TCR+ T cells. Furthermore, human DC which are active in MLR or in presenting antigens to primed-xcex1xcex2-T cells have not been shown to be capabl of presenting exogenous antigens for primary T cell activation.
The present invention relates to the isolation of human DC from the peripheral blood, their use as APC for the activation of primary and secondary xcex3xcex4-TCR+ T cell responses, and an in vitro method for assessing immune responsiveness of both unprimed and primed individuals to potentially immunogenic epitopes using DC as APC, and xcex3xcex4-TCR+ T cells as responders. Because DC are present at extremely low quantities in the human peripheral blood, their enrichment and purification are necessary in order to obtain adequate numbers for pulsing with antigens for the induction of both proliferative and cytotoxic xcex3xcex4-TCR+ T cell-mediated responses in vitro.
The invention is based, in part, on Applicants"" discovery that DC partially purified from human blood by sequential density gradient centrifugation function as potent APC for the sensitization of autologous naive xcex3xcex4-TCR+ T cells. As shown in the working example described herein in Example 7, infra, DC exposed to HIV envelope and gag peptides in vitro activate primary antigen-specific xcex3xcex4-TCR+ T cell cytotoxic responses, while similarly prepared autologous monocytes are not effective. The xcex3xcex4-T cell reactivity is not inhibited by antibodies to MHC class I and class II molecules, indicating that the T cells mediate cytotoxic activity in an MHC-unresricted manner. Additionally, xcex3xcex4-TCR+ T cells specific for Mycobacterium tuberculosis and for Staphylococcus aureus enterotoxin-A superantigen (SAE-A) have been generated using a similar procedure, and the T cells are shown to be capable of proliferating in response to the specific antigen. The anti-M. tuberculosis response is inhibited by antibodies to MHC class II determinants and to the CD1c molecule.
A wide variety of uses for this antigen presentation system is encompassed by the invention described herein, including but not limited to, the activation and expansion of antigen-specific xcex3xcex4-TCR+ T cells in vitro for use in adoptive cellular immunotherapy of infectious diseases and cancer, and the identification of antigenic epitopes for vaccine development. In particular, since the cytotoxic activity of the xcex3xcex4-T cells described herein is antigen specific but MHC unrestricted, these cells may be especially useful in immunotherapy without the need of matching the donor effector cells with the MHC of a recipient.